Haemophilia A is an inherited bleeding disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII) activity. The clinical manifestation is not on primary haemostasis—formation of the blood clot occurs normally—but the clot is unstable due to a lack of secondary thrombin formation. The disease is treated by intravenous injection of coagulation factor FVIII which is either isolated from blood or produced recombinantly. Haemophilia A patients with inhibitors may be treated by on-demand administration of factor Vila (FVIIa). Haemophilia B is caused by deficiency or dysfunction of coagulation factor IX (FIX) activity and patients can be treated by on demand administration of factor IX (FIX).
Current treatment recommendations are moving from traditional on-demand treatment towards prophylaxis. The circulatory half life of endogenous FVIII bound to von Willebrandt Factor is 12-14 hours and prophylactic treatment is thus to be performed several times a week in order to obtain a virtually symptom-free life for the patients. Circulatory half life of endogenous factor VII is less than 2 hours. Circulatory half life of endogenous factor IX is 19-24 hours. I.V. administration is for many, especially children and young persons, associated with significant inconvenience and/or pain. There is thus a need in the art for prolonging the circulatory half life of recombinant coagulation factors. There is likewise a need in the art for prolonging the half life of a number of other therapeutic proteins and peptides in a site-directed manner preferably resulting in a relatively homogenous and well defined product.
Various methods have been employed in the development of coagulation factors, and of pharmaceutical proteins in general, with significantly prolonged circulatory half life. A number of these methods relate to conjugation with hydrophilic polymers such as e.g. PEG (poly ethylene glycol). Conjugation with hydrophobic side chains do on the one hand constitute an attractive approach due to the fact that such groups might be completely degraded in vivo. On the other hand, conjugation of proteins with one or more well-defined hydrophobic moieties has so far not constituted an attractive approach for half-life prolongation of relatively large proteins, including coagulation factors such as e.g. FVIII. In fact, conjugation of such hydrophobic moieties to proteins would not be considered a realistic approach due to: 1) large proteins such as FVIII are only stable in aqueous buffers devoid of organic solvents; 2) lipophilic moieties such as fatty acids are insoluble in such aqueous buffers; 3) it would be doubtful that attachment of relatively small lipophilic moieties would result in any significant protraction of much larger proteins such as e.g. FVIII; 4) lipophilic moieties, when attached to the normally hydrophilic surface of proteins, may pertube their stability due to aggregation or energetically favorable but destructive unfolding; and 5) interaction of the protein with its natural binding partners may be pertubed in similar way leading to reduced biological activity.